This is a revised version of a competitive renewal of NIH grant 5R01DK033201-30. Since its inception, a major focus of this grant has been the study of the role of phosphorylation of insulin receptor substrates (IRS) and their downstream partners in insulin action and insulin resistance. Under this grant we have shown how different IRS proteins play differential roles in different insulin actions both in vivo and in vitro. We have identified and characterized at a molecular level a number of molecules and pathways that modify IRS signaling, including the stress kinases, SOCS proteins and the UPR. This work has led us to develop a new model of the insulin signaling network in which there are critical nodes which allow signal divergence into different pathways that provide complementary information to different downstream actions of insulin. These critical nodes also provide sites of positive and negative regulation of signaling and crosstalk between the insulin signaling pathway and other signaling pathways that can lead to alterations in insulin action in disease. The second major and growing focus of this grant has been to determine how genetic and acquired alterations in these signaling pathways can lead to insulin resistant states, such as type 2 diabetes and metabolic syndrome. Recently, we have focused on how small differences in either genes or environment can alter insulin action and diabetes risk. Using a combination of genetics, genomics and proteomics, we have shown how differences in expression of background genes in different strains of mice, such as protein kinase C (PKC) ?, can act as potent modifiers of insulin sensitivity between mouse strains. In very exciting recent studies using mice of three different genetic backgrounds, either commercially obtained or bred at Joslin, we have also shown how differences in environmental factors, including differences in the gut microbiome, can interact with genetic background and diet to contribute to important differences in development of obesity and insulin resistance between strains of mice. The specific aims for the next five years, moving forward progressively, are to: 1) continue to elucidate differences in IRS signaling complexes and their interacting downstream partners in different tissues using proteomic and phosphoproteomic approaches; 2) define the role and mechanisms by which background genes, especially PKC?, can modify insulin action at a molecular level including targets in the insulin signaling cascade and in the mitochondrion; and 3) using a new model system of mice with three different genetic backgrounds, either commercially obtained or environmentally conditioned at Joslin, determine how differences in the gut microbiome may also contribute to differences in insulin resistance and development of obesity and diabetes. The ultimate goal of this project is to define the critical nodes of regulation in the insulin signaling network in both normal and disease states and determine how genes and environment interact to create insulin resistance at a physiological and molecular level in obesity, diabetes and the metabolic syndrome.